This invention relates to oligonucleotide (ODN) based therapeutics, particularly the treatment of infections of the human immunodeficiency virus (HIV).
The present invention relates to ODNs suitable for use in treatment of HIV infected individuals by inhibition of replication of HIV in infected cells.
HIV is responsible for the disease that has come to be known as acquired immune deficiency syndrome (AIDS). Although initially recognized in 1981, no cure has yet been found for this inevitably fatal disease. HIV is spread by a variety of means such as sexual contact, infected blood or blood products and perinatally. Because of the complexity of HIV infection and the paucity of effective therapies, a great deal of effort has been expended in developing methods for detecting, treating and preventing infection. Diagnostic procedures have been developed for identifying infected persons, blood and other biological products.
The HIV genome has been well characterized. Its approximately 10 kb encode sequences containing regulatory segments for HIV replication as well as the gag, pol and env genes coding for the core proteins, the reverse transcriptase-protease-endonuclease, and the internal and external envelope glycoproteins, respectively. HIV tends to mutate at a high rate causing great genetic variation between strains of the viruses and indeed between virus particles of a single infected individual. There are a few xe2x80x9cconservedxe2x80x9d regions of the HIV genome which tend not to mutate. These regions are presumed to encode portions of proteins essential for virus function which can thus withstand very few mutational events.
The HIV env gene encodes the glycoprotein, gpl60, which is normally processed by proteolytic cleavage to form gp120, the external viral glycoprotein, and gp41, the viral transmembrane glycoprotein. The gpl120remains associated with HIV virions by virtue of noncovalent interactions with gp41. These noncovalent interactions are weak, consequently most of the gpl120 is released from cells and virions in a soluble form.
Like most viruses, HIV often elicits the production of neutralizing antibodies. Unlike many other viruses and other infectious agents for which infection leads to protective immunity, however, HIV specific antibodies are insufficient to halt the progression of the disease. Therefore, in the case of HIV, a vaccine that elicits the immunity of natural infection could prove to be ineffective. In fact, vaccines prepared from the HIV protein gp160 appear to provide little immunity to HIV infection although they elicit neutralizing antibodies. The failure to produce an effective anti-HIV vaccine has led to the prediction that an effective vaccine will not be available until the end of the 1990""s. Therapeutic agents currently used in treatment of AIDS often cause severe side-effects which preclude their use in many patients. It would, thus, be useful to have alternative methods of treating and preventing the disease that do not entail vaccination and currently available pharmaceutical agents.
Recently, attempts have been made to moderate protein production associated with viral infections by interfering with the mRNA molecules that direct their synthesis. By interfering with the production of proteins, it has been hoped to effect therapeutic results with maximum effect and minimal side effects. It is the general object of such a therapeutic approach to interfere with or otherwise modulate gene expression leading to undesired protein formation.
One method for inhibiting specific gene expression which is believed to have promise is the xe2x80x9cantisensexe2x80x9d approach. Single-stranded nucleic acid, primarily RNA, is the target molecule for ODNs that are used to inhibit gene expression by an antisense mechanism. A number of workers have reported such attempts: Stein and Cohen (1988) Cancer Res., 48:2659-2668; Walder (1988) Genes and Development, 2:502-504; Marcus-Sekura (1988) Anal. Biochem., 172:289-295; Zon (1987) J. Pro. Chem., 6:131-145; Zon (1988) Pharm. Res., 5:539-549; Van der Krol et al. (1988) Biotechniques, 6:958-973; and Loose-Mitchell (1988) TIPS, 9:45-47. Antisense ODNs are postulated to exert an effect on target gene expression by hybridizing with a complementary RNA sequence. The hybrid RNA-ODN duplex appears to interfere with one or more aspects of RNA metabolism including processing, translation and metabolic turnover. Chemically modified ODNs have been used to enhance nuclease stability and cell permeability.
Duplex DNA can be specifically recognized by oligomers based on a recognizable nucleomonomer sequence. The motif termed xe2x80x9cGTxe2x80x9d recognition has been described by Beal et al. (1992) Science, 25:1360-1363; Cooney et al. (1988) Science, 241:456-459; and Hogan et al., EP Publication 375408. In the G-T motif, the ODN is oriented antiparallel to the target purine-rich sequence and A-T pairs are recognized by adenine or thymine residues and G-C pairs by guanine residues.
Sequence-specific targeting of both single-stranded and duplex target sequences has applications in diagnosis, analysis, and therapy. Under some circumstances wherein such binding is to be effected, it is advantageous to stabilize the resulting duplex or triplex over long time periods.
Covalent crosslinking of the oligomer to the target provides one approach to prolong stabilization. Sequence-specific recognition of single-stranded DNA accompanied by covalent crosslinking has been reported by several groups. For example, Vlassov at al. (1986) Nuc. Acids Res., 14:4065-4076, describe covalent bonding of a single-stranded DNA fragment with alkylating derivatives of nucleomonomers complementary to target sequences. A report of similar work by the same group is that by Knorre et al. (1985) Biochimie, 67:785-789. It has also been shown that sequence-specific cleavage of single-stranded DNA can be mediated by incorporation of a modified nucleomonomer which is capable of activating cleavage. Iverson and Dervan (1987) J. Am. Chem. Soc., 109:1241-1243. Covalent crosslinking to a target nucleomonomer has also been effected using an alkylating agent complementary to the single-stranded target nucleomonomer sequence. Meyer et al. (1989) J. Am. Chem. Soc., 111:8517-8519. Photoactivated crosslinking to single-stranded ODNs mediated by psoralen has been disclosed. Lee et al. (1988) Biochem., 27:3197-3203. Use of crosslinking in triple-helix forming probes has also been disclosed. Horne et al. (1990) J. Am. Chem. Soc., 112:2435-2437.
Use of N4, N4-ethanocytosine as an alkylating agent to crosslink to single-stranded and double-stranded oligomers has also been described. Webb and Matteucci (1986) J. Am. Chem. Soc., 108:2764-2765; (1986) Nuc. Acids Res., 14:7661-7674; and Shaw et al. (1991) J. Am. Chem. Soc., 113:7765-7766. These papers also describe the synthesis of ODNs containing derivatized cytosine. The synthesis of oligomers containing N6, N6-ethanoadenine and the crosslinking properties of this residue in the context of an ODN binding to a single-stranded DNA has been described. Matteucci and Webb (1987) Tetrahedron Letters, 28:2469-2472.
In a recent paper, sequence-specific binding of an octathymidylate conjugated to a photoactivatable crosslinking agent to both single-stranded and double-stranded DNA is described. Praseuth et al. (1988) Proc. Natl. Acad. Sci. (USA), 85:1349-1353. In addition, targeting duplex DNA with an alkylating agent linked through a 5xe2x80x2-phosphate of an ODN has been described. Vlassov et al. (1988) Gene313-322; and Fedorova et al. (1988) FEBS Lett., 228:273-276.
In effecting binding to obtain a triplex, to provide for instances wherein purine residues are concentrated on one chain of the target and then on the. opposite chain, oligomers of inverted polarity can be provided. By xe2x80x9cinverted polarityxe2x80x9d is meant that the oligomer contains tandem sequences which have opposite polarity, i.e., one having polarity 5xe2x80x2xe2x86x923xe2x80x2 followed by another with polarity 3xe2x80x2xe2x86x925xe2x80x2, or vice versa. This implies that these sequences are joined by linkages which can be thought of as effectively a 3xe2x80x2xe2x80x943xe2x80x2 internucleoside junction (however the linkage is accomplished), or effectively a 5xe2x80x2xe2x80x945xe2x80x2 internucleoside junction. Such oligomers have been suggested as by-products of reactions to obtain cyclic ODNs. Capobionco et al. (1990) Nuc. Acids Res., 18:2661-2669. Compositions of xe2x80x9cparallel-stranded DNAxe2x80x9d designed to form hairpins secured with AT linkages using either a 3xe2x80x2xe2x80x943xe2x80x2 inversion or a 5xe2x80x2xe2x80x945xe2x80x2 inversion have been synthesized. van de Sande et al. (1988) Science, 241:551-557. In addition, triple helix formation using oligomers which.contain 3xe2x80x2xe2x80x943xe2x80x2 linkages have been described. Horne and Dervan (1990) J. Am. Chem. Soc., 112:2435-2437; and Froehler et al. (1992) Biochem., 31:1603-1609.
The use of triple helix (or triplex) complexes as a means for inhibition of the expression of target gene expression has been previously adduced (International Application No. PCT/US89/05769). Triple helix structures have been shown to interfere with target gene expression (International Application No. PCT/US91/09321; and Young et al. (1991) Proc. Natl. Acad. Sci., 88:10023-10026), demonstrating the feasibility of this approach.
Various modifications have been found to be suitable for use in ODNs. Oligomers containing 5-propynyl modified pyrimidines have been described. Froehler et al. (1992) Tetrahedron Letters, 33:5307-5310. 2xe2x80x2-Deoxy-7-deazaadenosine and 2xe2x80x2-deoxy-7-deazaguanosine have been incorporated into ODNs and assessed for binding to the complementary DNA sequences. Thermal denaturation analysis (Tm) has shown that these substitutions modestly decrease the Tm of the duplex when these analogs are substituted for 2xe2x80x2-deoxyadenosine and 2xe2x80x2-deoxyguanosine. Seela and Kehne (1987) Biochem., 26:2232-2238; and Seela and Driller (1986) Nuc. Acids Res., 14:2319-2332. It has also been shown that ODNs which alternate 2xe2x80x2-deoxy-7-deaza-adenosine and -thymidine can have a slightly enhanced duplex Tm over ODNs containing 2xe2x80x2-deoxy-adenosine and -thymidine. Seela and Kehne (1985) Biochem., 24:7556-7561.
2xe2x80x2, 3xe2x80x2-dideoxydeazapurine nucleosides have been used as chain terminators for DNA sequencing. 7-propargyl amino linkers are used for incorporation of fluorescent dyes into the nucleoside triphosphates
DNA synthesis via amidite and hydrogen phosphonate chemistries has been described. U.S. Pat. Nos. 4,725,677; 4,415,732; 4,458,066; and 4,959,463.
Prior attempts at antisense inhibition of HIV have focused on inhibition of the synthesis of some particular viral protein thought to be essential to the success of the infection and to RNAs which are believed to have important biological function. It has now been found that inhibition of viral gene expression and replication can be more efficiently achieved by targeting the conserved sites of the viral RNAs that signal the synthesis of conserved HIV proteins, particularly the p24 core antigen protein.
The present invention is directed to ODNs comprising nucleotide sequences sufficiently complementary to conserved regions of human immunodeficiency virus genetic material such that when bound to said region, the ODNs effectively prevent expression of the genetic material.